Typically, a diagnostic method using a short nucleic acid probe (generally, DNA or PNA) having a single chain has been used for many purposes, such as for checking or detecting disease causing-genes, mutations, infectious agents (for example, viruses, microorganisms, and the like), genotyping, gene expression, and the like. Generally, the short nucleic acid probe has been used while being fixed on various solid surfaces including a glass plate surface, a metal plate surface, and a bead surface. By way of example, one of advantages of a DNA micro array (or a DNA chip) using a short nucleic acid probe is that many different target nucleic acid sequences can be analyzed at the same time. Such a technique has been used in hospitals and clinics for diagnosis, and other various techniques have been combined and optimized for easy use and/or reliable determination for clinical diagnoses.
Typically, the diagnostic method using a short nucleic acid probe requires a long process as follows: (a) extracting a nucleic acid (DNA or RNA) from a biological sample (for example, tissue, a cell, blood, serum, a body fluid, and the like); (b) amplifying a target nucleic acid; and (c) finally, hybridizing the amplified nucleic acid sequence with a short nucleic acid probe fixed on a solid (for example, glass slide or bead) surface. Herein, in order to detect a RNA sequence, a RNA needs to be converted into a DNA by means of a reverse transcription reaction prior to the amplification.
As can be seen from the typical process required for detecting a target nucleic acid, a molecular biological diagnostic method using a short nucleic acid probe requires a lot of time and effort and it is inconvenient. Therefore, a simple process is required for conveniently and economically analyzing a nucleic acid sequence with high throughput, and it is important, particularly, for clinical diagnoses in hospitals and clinics.
Further, sensitivity of a method used for detecting a hybridized product is important. Detection sensitivity is important, particularly, in detecting infectious agents (for example, viruses, microorganisms, and the like) and also important for prognosis of treatment. By way of example, the success or failure of treatment for infectious agents can be determined by the number of residual infectious agents remaining in human body fluids (for example, blood). As another example, the success or failure of cancer treatment can be determined by existence of a cancer causing gene (i.e. existence of a cancer cell).
Typically, it has been known that detection sensitivity of a target nucleic acid sequence using a short nucleic acid probe fixed on a solid surface is lower several times than sensitivity of a real-time PCR. Therefore, a method for improving detection sensitivity using a short nucleic acid probe and desirably, a detection method with improved sensitivity capable of detecting a single target molecule need to be developed.
There has been used a diagnostic method in which a branched DNA connected with multiple labels is synthesized without amplifying a diagnosis target nucleic acid and a high signal and sensitivity thereof can be obtained through a bipartite oligonucleotide probe, i.e. an oligonucleotide that connects the branched DNA with the diagnosis target nucleic acid combined with a capture probe on a solid surface by means of hybridization. However, according to this method, the branched DNA needs to be synthesized outside and the bipartite oligonucleotide probe that recognizes a diagnosis target as well as the capture probe fixed on the solid surface needs to be synthesized. Although this method has high sensitivity, it requires a lot of time and it is inconvenient to perform many processes.